Genotyping Of Human Papillomavirus By A New Luminex XMAP DNA Liquid Chip In Women Of High-Risk Group Of Shandong Province

Objective To evaluate the sensitivity, specificity and the diagnostic applicability of HPV liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong Province.Methods1. Sensitivity and specificity of Luminex XMAP DNA Liquid ChipBased on the newest HPV genome sequence published by GeneBank, we prepare the HPV control plasmids quantitatively. Under the accurate and optimized condition, we repeat the detection of standard preparation plasmids at four different concentrations (5 copies/5ul,10 copies/5ul,20 copies/5ul, and 50 copies/5ul, having been got through accurate quantification and calculation) to assure the sensitivity of Luminex XMAP Liquid Chip. To assure the spesitifity of Luminex XMAP Liquid Chip with normal control genomic human DNA.2. The evaluation of the diagnostic applicability of Luminex XMAP DNA Liquid ChipDetect HPV DNA with both the liquid chip which can simultaneously detect and identify 26 common HPV genotypes and Hybrid Capture II in a total of 264 cases, including 231 cases of the Formalin-fixed, paraffin-embedded tissue specimens(FFPE) and 33 cases of cervical scrapes. The accordance of two methods was compared and DNA sequencing was performed in all the conflicting samples and all the single infection samples to evaluate the accuracy of the liquid chip.3. Genotyping of HPV by Luminex XMAP DNA Liquid Chip in women of high-risk group of Shandong ProvinceDetect HPV genotypes with the liquid chip in a total of 2,925 cervical scrapes obtained from gynecological outpatients. Among 639 cases also performing pathology cytologic and histologic diagnoses, 184 cases are in group of normal cytology,266 cases are in group of,77 cases are in group of CIN I,7 cases are in group of CIN I- II, 46 cases are in group of CIN I- II, 46 cases are in group of CIN I- II and 13 cases are in group of cervical cancer. Analyse the relationship between HPV types and different cervical diseases by the distribution of HPV genotypes and pathology diagnosis.Results1. The data of the specificity and sensitivity of our liquid chip on PCR amplicons was generated from HPV control plasmids; Results of the tests at the concentration of 10 copies /5ul, 20 copies /5ul and 50 copies /5u are all positive; the results of the six parallel wells at the concentration of 5 copies/5ul showed that only HPV16 and HPV18 were all positive for the six wells, but for other 24 types' tests, at least one of the 6 parallel wells is negative, so that the detection limit for type-specific HPV testing is 10 copies /5ul using the liquid chip.Analysis of these data showed that our liquid chip perfectly discriminated all targets. The results of HPV testing on normal control genomic human DNA by Luminex XMAP Liquid Chip are negative.The specificity and sensitivity of liquid chip are necessarily sufficient.2. There is a excellent agreement between the methods (kappa =0.8889, P < 0.001), the Kappa value are 1,0.8372,0.6411 and 0.6540 for normal cytology, CIN II, CIN III, and carcinomas specimens, respectively. The concordance of DNA sequencing and liquid chip on all the single types is 98.29%.3. The overall incidence of HPV in our samples was 36.0 %( 1,054/2,925), 23 types of all 26 types on liquid chip were found. The most common genotypes found were HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18(8.87%)and HPV-11(6.94%). Among all the positive types, 87.32% were high-risk HPV genotypes and 13.68% were low-risk HPV genotypes. Both single and multiple types were easily identified, showing 66.22% (n = 698) single type and 33.78% (n = 356) multiple types. Of all the 1054 HPV-positive cases, 261(24.8%) was occupied by women 21 to 25 years of age and was progressively lower by older age groups, reaching 4.9% by women 51 to 65 years old. The incidence of HPV in our samples was 23.37%, 33.08%, 54.54%,57.14%,82.61%,91.30%and 100% for normal cytology, inflammation, CIN I, CIN I- II, CIN II, CIN III, and carcinomas specimens, respectively. Infections with more that one virus were common and accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I-II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II ,III were 88.57%, 76.63%, 68.89% and 92.16% respectively.Conclusions1. It is the first time to detect HPV genotypes by the Luminex XMAP liquid chip at home, and to evaluate the sensitivity, specificity and clinical validity of the liquid chip firstly.2. It is the first time to evaluate the infection of HPVand the most common HPV genotypes in women of Shandong, which could conduce to diagnose the ceivix disease and vaccination studies.3. The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31.The HPV-positive rate and the multiple infections both increased along with the increase of the grade of cervical lesions. There were more young women than older women among all the HPV-positive women.4. With the sufficient sensitivity and speciticity, multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.